Monocyte Trafficking Research Articles

Lipoprotein(a) induces spontaneous aortic dissection via increasing monocyte mediated vascular smooth muscle cell phenotype transformation in human Lp(a) transgenic mice

Abstract Background Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent risk factor of atherosclerotic cardiovascular disease and aortic valve stenosis. Recent studies have reported that high levels of Lp(a) are strongly associated with aortic dissection (AD), independent of other cardiovascular risk factors. However, orthologues of Lp(a) are only found in humans, Old World monkeys, apes, and hedgehogs. The lack of a proper animal model has hindered elucidation of the true pathophysiological mechanisms of Lp(a). Here, we conducted the first humanized Lp(a) transgenic mice experiment to verify the pathological mechanism of Lp(a) in aortic dissection. Methods We conducted ApoE knock out and human ApoA transgenic mice via CRISPR-Cas9 and then crossed with human ApoB transgenic mice to achieve high levels of human-like Lp(a) particles in plasma. Plasma was assayed by ELISA for the concentrations of each lipoprotein. Echocardiography was performed to detect aortic morphology in vivo. Single cell transcriptome analysis was performed on the dissection lesion. Cell interaction was examined in macrophage from Lp(a) transgenic mice and vascular smooth muscle cell (VSMC) coculture system. Result Our results indicate that the concentration of ApoA and ApoB in plasma were increased in transgenic mice fed a chow diet. Low density lipoprotein (LDL) levels were increased and shows that LDL is the major cholesterol carrier in these mice. We were surprised to find that the dissection of the ascending aortic arch could be seen by echocardiography in transgenic mice as early as 2 months, and transgenic mice showed significant increase in aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions, resulting in a survival rate of only 58% after 12 months. Single cell transcriptome analysis revealed that Lp(a) transgene induced the inflammatory response, dedifferentiation and pathological VSMC phenotypical switch, and matrix metalloproteinase expression in macrophages. Monocytes isolated from transgenic mice with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce proinflammatory cytokines on stimulation. Monocytes isolated from wild type mice cultured in media with high level Lp(a) for 24h also showed the ability to facilitated cocultured VSMC phenotype transformation, which was reversed by E06, a monoclonal antibody blocking the oxidized phospholipids in Lp(a). Conclusion These findings demonstrate that Lp(a) induces spontaneous aortic dissection via increasing monocyte trafficking to the arterial wall, mediating proinflammatory responses and VSMC phenotype transformation through its OxPL content. The study presents a rodent model of spontaneous aortic dissection and provide a novel mechanism by which Lp(a) mediates aortic dissection.Graphical abstract

Immunometabolic cues recompose and reprogram the microenvironment around implanted biomaterials.

Circulating monocytes infiltrate and coordinate immune responses in tissues surrounding implanted biomaterials and in other inflamed tissues. Here we show that immunometabolic cues in the biomaterial microenvironment govern the trafficking of immune cells, including neutrophils and monocytes, in a manner dependent on the chemokine receptor 2 (CCR2) and the C-X3-C motif chemokine receptor 1 (CX3CR1). This affects the composition and activation states of macrophage and dendritic cell populations, ultimately orchestrating the relative composition of pro-inflammatory, transitory and anti-inflammatory CCR2+, CX3CR1+ and CCR2+ CX3CR1+ immune cell populations. In amorphous polylactide implants, modifying immunometabolism by glycolytic inhibition drives a pro-regenerative microenvironment principally by myeloid cells. In crystalline polylactide implants, together with arginase-1-expressing myeloid cells, T helper 2 cells and γδ+ T cells producing interleukin-4 substantially contribute to shaping the metabolically reprogrammed pro-regenerative microenvironment. Our findings inform the premise that local metabolic states regulate inflammatory processes in the biomaterial microenvironment.

Infiltrating monocytes drive cardiac dysfunction in a cardiomyocyte-restricted mouse model of SARS-CoV-2 infection.

Cardiovascular manifestations of coronavirus disease 2019 (COVID-19) include myocardial injury, heart failure, and myocarditis and are associated with long-term disability and mortality. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antigens are found in the myocardium of COVID-19 patients, and human cardiomyocytes are susceptible to infection in cell or organoid cultures. While these observations raise the possibility that cardiomyocyte infection may contribute to the cardiac sequelae of COVID-19, a causal relationship between cardiomyocyte infection and myocardial dysfunction and pathology has not been established. Here, we generated a mouse model of cardiomyocyte-restricted infection by selectively expressing human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-2 receptor, in cardiomyocytes. Inoculation of Myh6-Cre Rosa26loxP-STOP-loxP-hACE2 mice with an ancestral, non-mouse-adapted strain of SARS-CoV-2 resulted in viral replication within the heart, accumulation of macrophages, and moderate left ventricular (LV) systolic dysfunction. Cardiac pathology in this model was transient and resolved with viral clearance. Blockade of monocyte trafficking reduced macrophage accumulation, suppressed the development of LV systolic dysfunction, and promoted viral clearance in the heart. These findings establish a mouse model of SARS-CoV-2 cardiomyocyte infection that recapitulates features of cardiac dysfunctions of COVID-19 and suggests that both viral replication and resultant innate immune responses contribute to cardiac pathology.IMPORTANCEHeart involvement after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection occurs in multiple ways and is associated with worse outcomes in coronavirus disease 2019 (COVID-19) patients. It remains unclear if cardiac disease is driven by primary infection of the heart or immune response to the virus. SARS-CoV-2 is capable of entering contractile cells of the heart in a culture dish. However, it remains unclear how such infection affects the function of the heart in the body. Here, we designed a mouse in which only heart muscle cells can be infected with a SARS-CoV-2 strain to study cardiac infection in isolation from other organ systems. In our model, infected mice show viral infection, worse function, and accumulation of immune cells in the heart. A subset of immune cells facilitates such worsening heart function. As this model shows features similar to those observed in patients, it may be useful for understanding the heart disease that occurs as a part of COVID-19.

Patient-specific vascularized tumor model: Blocking monocyte recruitment with multispecific antibodies targeting CCR2 and CSF-1R

Tumor-associated inflammation drives cancer progression and therapy resistance, often linked to the infiltration of monocyte-derived tumor-associated macrophages (TAMs), which are associated with poor prognosis in various cancers. To advance immunotherapies, testing on immunocompetent pre-clinical models of human tissue is crucial. We have developed an in vitro model of microvascular networks with tumor spheroids or patient tissues to assess monocyte trafficking into tumors and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via CCL7 and CCL2, mediated by CSF-1R. Additionally, a multispecific antibody targeting CSF-1R, CCR2, and neutralizing TGF-β (CSF1R/CCR2/TGF-β Ab) repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and blocks monocyte migration. This antibody also inhibits monocyte recruitment in patient-specific vascularized tumor models. In summary, this vascularized tumor model recapitulates the monocyte recruitment cascade, enabling functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment.

Innate immune memory after brain injury drives inflammatory cardiac dysfunction

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1β was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1β or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1β-mediated comorbidities, offering a framework for secondary prevention immunotherapy.

PI3Kγ inhibition circumvents inflammation and vascular leak in SARS-CoV-2 and other infections.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Rare damaging CCR2 variants are associated with lower lifetime cardiovascular risk.

Previous work has shown a role of CCL2, a key chemokine governing monocyte trafficking, in atherosclerosis. However, it remains unknown whether targeting CCR2, the cognate receptor of CCL2, provides protection against human atherosclerotic cardiovascular disease. Computationally predicted damaging or loss-of-function (REVEL>0.5) variants within CCR2 were detected in whole-exome-sequencing data from 454,775 UK Biobank participants and tested for association with cardiovascular endpoints in gene-burden tests. Given the key role of CCR2 in monocyte mobilization, variants associated with lower monocyte count were prioritized for experimental validation. The response to CCL2 of human cells transfected with these variants was tested in migration and cAMP assays. Validated damaging variants were tested for association with cardiovascular endpoints, atherosclerosis burden, and vascular risk factors. Significant associations were replicated in six independent datasets (n=1,062,595). Carriers of 45 predicted damaging or loss-of-function CCR2 variants (n=787 individuals) were at lower risk of myocardial infarction and coronary artery disease. One of these variants (M249K, n=585, 0.15% of European ancestry individuals) was associated with lower monocyte count and with both decreased downstream signaling and chemoattraction in response to CCL2. While M249K showed no association with conventional vascular risk factors, it was consistently associated with a lower risk of myocardial infarction (Odds Ratio [OR]: 0.66 95% Confidence Interval [CI]: 0.54-0.81, p=6.1×10-5) and coronary artery disease (OR: 0.74 95%CI: 0.63-0.87, p=2.9×10-4) in the UK Biobank and in six replication cohorts. In a phenome-wide association study, there was no evidence of a higher risk of infections among M249K carriers. Carriers of an experimentally confirmed damaging CCR2 variant are at a lower lifetime risk of myocardial infarction and coronary artery disease without carrying a higher risk of infections. Our findings provide genetic support for the translational potential of CCR2-targeting as an atheroprotective approach.

Ly6Chi Monocytes Are Metabolically Reprogrammed in the Blood during Inflammatory Stimulation and Require Intact OxPhos for Chemotaxis and Monocyte to Macrophage Differentiation.

Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a coordinated and precise manner. Here, we investigate the origin and transcriptional reprogramming of monocytes using a model of acute inflammation, zymosan-induced peritonitis. Monocyte trafficking and adoptive transfer experiments confirmed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single-cell transcriptomics revealed significant heterogeneity within the surface marker-defined CD11b+Ly6G-Ly6Chi monocyte populations within the blood and at the site of inflammation. We show that two major transcriptional reprogramming events occur during the initial six hours of Ly6Chi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events. Experimentally, we demonstrate that OxPhos via the intact mitochondrial electron transport chain is essential for murine and human monocyte chemotaxis. Moreover, OxPhos is needed for monocyte-to-macrophage differentiation and macrophage M(IL-4) polarisation. These new findings from transcriptional profiling open up the possibility that shifting monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.

Removal of the endothelial surface layer via hyaluronidase does not modulate monocyte and neutrophil interactions with the glomerular endothelium.

The endothelial surface layer (ESL), a layer of macromolecules on the surface of endothelial cells, can both impede and facilitate leukocyte recruitment. However, its role in monocyte and neutrophil recruitment in glomerular capillaries is unknown. We used multiphoton intravital microscopy to examine monocyte and neutrophil behavior in the glomerulus following ESL disruption with hyaluronidase. Constitutive retention and migration of monocytes and neutrophils within the glomerular microvasculature was unaltered by hyaluronidase. Consistent with this, inhibition of the hyaluronan-binding molecule CD44 also failed to modulate glomerular trafficking of these immune cells. To investigate the contribution of the ESL during acute inflammation, we induced glomerulonephritis via in situ immune complex deposition. This resulted in increases in glomerular retention of monocytes and neutrophils but did not induce marked reduction in the glomerular ESL. Furthermore, hyaluronidase treatment did not modify the prolonged retention of monocytes and neutrophils in the acutely inflamed glomerular microvasculature. These observations indicate that, despite evidence that the ESL has the capacity to inhibit leukocyte-endothelial cell interactions while also containing adhesive ligands for immune cells, neither of these functions modulate trafficking of monocytes and neutrophils in steady-state or acutely-inflamed glomeruli.

GTS-21 alleviates murine collagen-induced arthritis through inhibition of peripheral monocyte trafficking into the synovium

Emerging preclinical and clinical evidence reveals a critical role for the cholinergic anti-inflammatory pathway (CAP) in mediating rheumatoid arthritis (RA). Activation of CAP via vagus nerve stimulation or alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonists has previously been shown to significantly reduce inflammation and improve outcomes in animal models of experimental arthritis. In this study, we sought to determine the protective mechanism of CAP on inflammatory arthritis, specifically RA, by using a selective α7nAChR agonist, GTS-21, to examine the role of CAP in the recruitment of monocytes/macrophages into the synovium in a collagen-induced arthritis (CIA) mouse model.We found that GTS-21 ameliorated systemic and local synovial inflammation, thereby reducing synovial macrophage infiltration in CIA mice. Using in vivo imaging, we further demonstrated that GTS-21 suppressed the trafficking of monocytes into inflamed joints, while our in vitro Transwell assay data confirmed that GTS-21 reduced the migratory ability of monocytes. In addition, we found that GTS-21 reduced the number of peripheral inflammatory monocytes and down-regulated expression of the chemokines CCR2 and CCR5 on monocytes and CCL2 in the paw tissue. GTS-21 also mediated the expression levels of the adhesion molecules LFA-1 and VLA-4 on monocytes and VCAM-1 in the paw tissue, thereby blocking monocyte adhesion to the extracellular matrix.Together, our data demonstrate that GTS-21 alleviates arthritis by inhibiting peripheral monocyte trafficking into the synovium. Our findings describe a novel mechanism through which the cholinergic signaling pathway can reduce synovial inflammation in RA patients.

Cerebrospinal fluid CD14++CD16+monocytes in HIV-1 subtype C compared with subtype B.

CD14++CD16+ monocytes are susceptible to HIV-1 infection, and cross the blood-brain barrier. HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared to HIV-1B, which might influence monocyte trafficking into the CNS. We hypothesized that the proportion of monocytes in CSF in HIV-1C is lower than HIV-1B group. We sought to assess differences in monocyte proportions in cerebrospinal fluid (CSF) and peripheral blood (PB) between people with HIV (PWH) and without HIV (PWoH), and by HIV-1B and -C subtypes. Immunophenotyping was performed by flow cytometry, monocytes were analyzed within CD45 + and CD64 + gated regions and classified in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14lowCD16+). Among PWH, the median [IQR] CD4 nadir was 219 [32-531] cell/mm3; plasma HIV RNA (log10) was 1.60 [1.60-3.21], and 68% were on antiretroviral therapy (ART). Participants with HIV-1C and -B were comparable in terms of age, duration of infection, CD4 nadir, plasma HIV RNA, and ART. The proportion of CSF CD14++CD16+ monocytes was higher in participants with HIV-1C than those with HIV-1B [2.00(0.00-2.80) vs. 0.00(0.00-0.60) respectively, p = 0.03 after BH correction p = 0.10]. Despite viral suppression, the proportion of total monocytes in PB increased in PWH, due to the increase in CD14++CD16+ and CD14lowCD16+ monocytes. The HIV-1C Tat substitution (C30S31) did not interfere with the migration of CD14++CD16+ monocytes to the CNS. This is the first study to evaluate these monocytes in the CSF and PB and compare their proportions according to HIV subtype.

Abstract 380: Adult But Not Neonatal Platelets Promotes Monocyte Trafficking In Neonatal Mice

Background: Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there is little clinical data to support this practice and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune related mRNA compared to adult platelets. In this study we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications. Methods: Platelets and bone marrow isolated monocytes were co-cultured and monocyte phenotypes determined by RNA-seq and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet deficient thrombopoietin receptor mutant mice (TPOR -/- ) were transfused with adult or P7 platelets and monocyte phenotypes and trafficking were determined. Results: Adult and neonatal platelets had differential immune molecule expression, including Selp (P-selectin). Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6C hi ), but different trafficking phenotypes, as defined by CCR2 and CCR5 surface expression. Blocking P-selectin-PSGL-1 interaction, monocytes limited the adult platelet induced monocyte trafficking phenotype, as well as adult platelet induced monocyte migration in vitro . Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or P7 platelets; adult platelets increased monocyte CCR2 and CCR5 as well as monocyte chemokine migration, while P7 platelets did not. Conclusion: These data provide comparative insights into adult and neonatal platelet transfusion regulated monocyte functions.

Transfusion of Adult, but Not Neonatal, Platelets Promotes Monocyte Trafficking in Neonatal Mice.

Thrombocytopenia is common in preterm neonates. Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there are little clinical data to support this practice, and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune-related mRNA compared with adult platelets. In this study, we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications. Using RNA sequencing of postnatal day 7 and adult platelets, we determined age-dependent platelet gene expression. Platelets and naive bone marrow-isolated monocytes were cocultured and monocyte phenotypes determined by RNA sequencing and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet-deficient TPOR (thrombopoietin receptor) mutant mice were transfused with adult or postnatal day 7 platelets and monocyte phenotypes and trafficking were determined. Adult and neonatal platelets had differential immune molecule expression, including Selp. Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6Chi) but different trafficking phenotypes, as defined by CCR2 and CCR5 mRNA and surface expression. Blocking P-sel (P-selectin) interactions with its PSGL-1 (P-sel glycoprotein ligand-1) receptor on monocytes limited the adult platelet-induced monocyte trafficking phenotype, as well as adult platelet-induced monocyte migration in vitro. Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or postnatal day 7 platelets; adult platelets increased monocyte CCR2 and CCR5, as well as monocyte chemokine migration, whereas postnatal day 7 platelets did not. These data provide comparative insights into adult and neonatal platelet transfusion-regulated monocyte functions. The transfusion of adult platelets to neonatal mice was associated with an acute inflammatory and trafficking monocyte phenotype that was platelet P-sel dependent and may have an impact on complications associated with neonatal platelet transfusions.

Gallate Moiety of Catechin Is Essential for Inhibiting CCL2 Chemokine-Mediated Monocyte Recruitment

Leukocyte recruitment witnesses an orchestrated complex formation between the chemokines and their molecular partners. CCL2 chemokine that regulates monocyte trafficking is a worthwhile system from the pharmaceutical perspective. In the current study, four major catechins (EC/EGC/ECG/EGCG) were assessed for their inhibitory potential against CCL2-regulated monocyte/macrophage recruitment. Interestingly, catechins with the gallate moiety (ECG/EGCG) could only attenuate the CCL2-induced macrophage migration. These molecules specifically bound to CCL2 on a pocket comprising the N-terminal, β0-sheets, and β3-sheets, and the binding affinity of ECGC (Kd = 22 ± 4 μM) is ∼4 times higher than that of the ECG complex (Kd = 85 ± 6 μM). MD simulation analysis evidenced that the molecular specificity/stability of CCL2-catechin complexes is regulated by multiple factors, including stereospecificity, number of hydroxyl groups on the annular ring-B, the positioning of the carbonyl group, and the methylation of the galloyl ring. Further, a significant overlap on the binding surface of CCL2 for EGCG/ECG and receptor interactions as evidenced from NMR data provided the rationale for the observed inhibition of macrophage migration in response to EGCG/ECG binding. In summary, these galloylated epicatechins can be considered as potent protein-protein interaction (PPI) inhibitors that regulate CCL2-directed leukocyte recruitment for resolving inflammatory/immunomodulatory disorders.

Endothelial cell CD36 mediates stroke-induced brain injury via BBB dysfunction and monocyte infiltration in normal and obese conditions

CD36 expressed in multiple cell types regulates inflammation, vascular function, and innate immunity. Specifically, CD36 in microvascular endothelial cells (ECs) signals to elicit inflammation and causes EC death. This study investigated roles for EC-CD36 on acute stroke pathology in normal and obese conditions. Obesity induced by a high-fat diet (HD) selectively increased CD36 expression in ECs, not in monocytes/macrophages, in the post-ischemic brain. Mice deficient CD36 in ECs (ECCD36−/−) showed reduced injury size and vascular permeability in normal conditions. While control mice fed a HD developed obesity and aggravated stroke injury, ECCD36−/− mice were resistant to develop an obesity phenotype. Subjecting ECCD36−/− mice to stroke resulted in reduced injury size and BBB disruption. Moreover, the mice had reduced MCP-1 and CCR2 gene expression, resulting in reduced monocyte trafficking with improved survival and acute motor function. Reduced MCP-1 and CCR2 expression was still evident in ECCD36−/− mice subjected to severe stroke, suggesting that monocyte trafficking is an infarct-independent metabolic effect associated with specific EC-CD36 deletion. Our findings demonstrate the importance of EC-CD36 in developing vascular comorbidities and suggest that targeting EC-CD36 is a potential preventative strategy to normalize vascular risk factors, leading to improved acute stroke outcomes.

Malnutrition-related parasite dissemination from the skin in visceral leishmaniasis is driven by PGE2-mediated amplification of CCR7-related trafficking of infected inflammatory monocytes.

People are infected with Leishmania donovani when the parasite is deposited in the dermis during the blood meal of the sand fly vector. Most infected people develop a subclinical latent infection, but some develop progressive visceral leishmaniasis. Malnutrition is a risk factor for the development of active VL. We previously demonstrated increased parasite dissemination from the skin to visceral organs in a murine model of malnutrition. Here we investigated the mechanism of early parasite dissemination. After delivery of L. donovani to the skin, we found enhanced capture of parasites by inflammatory monocytes and neutrophils in the skin of malnourished mice. However, parasite dissemination in malnourished mice was driven primarily by infected inflammatory monocytes, which showed increased CCR7 expression, greater intrinsic migratory capacity, and increased trafficking from skin to spleen. PGE2 production, which was increased at the site of skin infection, increased monocyte CCR7 expression and promoted CCR7-related monocyte-mediated early parasite dissemination in malnourished mice. Parasite dissemination in monocytes was reduced by inhibition of PGE2, knockdown or silencing of CCR7 in monocytes, and depletion of inflammatory monocytes through administration of diphtheria toxin to CSFR1-DTR transgenic mice that have monocyte-specific DT receptor expression. CCR7-driven trafficking of infected inflammatory monocytes through the lymph node was accompanied by increased expression of its ligands CCL19 and CCL21. These results show that the CCR7/PGE2 axis is responsible for the increased trafficking of L. donovani-infected inflammatory monocytes from the skin to the spleen in the malnourished host. Undernutrition and production of PGE2 are potential targets to reduce the risk of people developing VL. Nutritional interventions that target improved immune function and reduced PGE2 synthesis should be studied in people at risk of developing VL.

Heterozygous premature termination in zinc-finger domain of Krüppel-like factor 2 gene associates with dysregulated immunity.

Krüppel-like factor 2 (KLF2) is a transcription factor with significant roles in development, maturation, differentiation, and proliferation of several cell types. In immune cells, KLF2 regulates maturation and trafficking of lymphocytes and monocytes. KLF2 participates in regulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Although pulmonary arterial hypertension (PAH) related to KLF2 genetic variant has been suggested, genetic role of KLF2 associated with immune dysregulation has not been described. We identified a family whose members suffered from lymphopenia, autoimmunity, and malignancy. Whole exome sequencing revealed a KLF2 p.(Glu318Argfs*87) mutation disrupting the highly conserved zinc finger domain. We show a reduced amount of KLF2 protein, defective nuclear localization and altered protein-protein interactome. The phenotypically variable positive cases presented with B and T cell lymphopenia and abnormalities in B and T cell maturation including low naive T cell counts and low CD27+IgD-IgM- switched memory B cells. KLF2 target gene (CD62L) expression was affected. Although the percentage of (CD25+FOXP3+, CD25+CD127-) regulatory T cells (Treg) was high, the naive Treg cells (CD45RA+) were absent. Serum IgG1 levels were low and findings in one case were consistent with common variable immunodeficiency (CVID). Transcription of NF-κβ pathway genes and p65/RelA phosphorylation were not significantly affected. Inflammasome activity, transcription of genes related with JAK/STAT pathway and interferon signature were also comparable to controls. Evidence of PAH was not found. In conclusion, KLF2 variant may be associated with familial immune dysregulation. Although the KLF2 deficient family members in our study suffered from lymphopenia, autoimmunity or malignancy, additional study cohorts are required to confirm our observations.

Antiplatelet Effects of Inhibiting Coagulation Factor XI in a Diet-Induced Obesity Model of Early Atherosclerosis

Background: Atherosclerosis-associated cardiovascular disease (CVD) remains the leading cause of non-communicable death worldwide. In the setting of atherosclerosis, platelets facilitate monocyte trafficking and drive inflammatory pathways. Previous studies have shown that coagulation factor (F) XI can interact directly with multiple pathways that are complicit in CVD, including platelet activation. Therefore, the objective of this study was to investigate the effects of pharmacologically inhibiting FXI on systemic platelet function in a diet-induced obesity model of early atherosclerosis. Methods: Five rhesus macaques on a high-fat diet for ≥2 years with detectable carotid intimal thickening were administered a FXI function-blocking antibody, h1A6 (2 mg/kg). Blood was collected at baseline, one, and seven days after treatment. Flow cytometry studies were performed to asses platelet function in lean rhesus macaques on a standard chow diet, as well as obese rhesus macaques a high fat diet before and after treatment with h1A6. Platelet reactivity and platelet-leukocyte aggregate (PLA) formation in response to platelet hemostatic agents against GPVI (collagen-related peptide, CRP-XL) and PAR-1 (thrombin analog, TRAP-6) were measured by P-selectin expression. The coagulability of platelets in response to CRP-XL or TRAP-6 was measured by phosphatidylserine (PS) exposure. Results: Obese NHPs demonstrated increased P-selectin expression and phosphatidylserine exposure in response to GPVI and PAR-1 agonists relative to lean NHPs. This suggests that diet-induced obesity may increase platelet sensitivity, PLA formation, and platelet coagulability. Administration of a FXI function-blocking antibody resulted in prolonged clotting times, decreased GPVI- and PAR-1 mediated platelet sensitivity, and reduced PLA formation compared to basal levels. Phosphatidyl serine exposure was not affected by FXI inhibition. Conclusions: Pharmacological inhibition of FXI reversed the elevated platelet sensitivity and PLA formation observed in the diet-induced obesity model. Taken together, this suggests that coagulation FXI may modulate the exacerbated platelet function that is associated with atherosclerosis.

IMMU-23. ELIMINATING MONOCYTE CHEMOATTRACTION INVOKES COMPENSATORY NEUTROPHIL INFLUX AND PRONEURAL TO MESENCHYMAL TRANSITION IN GLIOBLASTOMA

Abstract Myeloid cells comprise the majority of immune cells in tumors, where their content and composition is determined by tumor type and driver mutation. While these cells are essential for shaping the tumor microenvironment, promoting tumor growth, and contributing to therapeutic resistance, targeting tumor-associated myeloid cells, including bone-marrow-derived monocytes and neutrophils, has not been successful in the clinics. Monocyte chemoattractant protein (MCP) family, comprising of Ccl2, Ccl7, Ccl8, Ccl12, are essential for monocytes trafficking to the tumor sites. To eliminate monocyte recruitment, we leveraged CRISPR/Cas-9 based gene editing tool to generate a mouse strain that is devoid of all MCP genes, which we termed quadruple MCP knockout (qMCP-/-). Using these mice in combination with genetically engineered mouse models (GEMM) of glioblastoma (GBM), we abolished tumor monocyte infiltration. Due to the functional redundancy of MCP family members, we show that targeting individual MCP genes leads to compensation by other MCPs. In contrast, when all MCPs are genetically deleted and monocyte recruitment is abolished, neutrophil infiltration ensues. Single-cell RNA sequencing revealed that intratumoral neutrophils promoted proneural-to-mesenchymal transition in GBM, and supported tumor aggression by facilitating hypoxia response via TNF production. Remarkably, pharmacologic or genetic interventions that suppress both monocytes and neutrophil infiltration improve the survival of GBM-bearing mice. Taken together, our findings establish that specific subsets of myeloid cells can influence the dynamism of tumor microenvironment, and they emphasize the importance of targeting both monocytes and neutrophils simultaneously for effective GBM immunotherapy.

Immune Modifying Effect of Drug Free Biodegradable Nanoparticles on Disease Course of Experimental Autoimmune Neuritis.

Guillain-Barre syndrome (GBS) is an autoimmune disease of demyelination and inflammation of peripheral nerves. Current treatments are limited to plasma exchange and intravenous immunoglobulins. Cargo-free nanoparticles (NPs) have been evaluated here for their therapeutic benefit on the disease course of experimental autoimmune neuritis (EAN), mimicking the human GBS. NPs prepared from poly-lactic co-glycolic acid (PLGA) with variable size and surface charge (i.e., 500 nm vs. 130 nm, polyvinyl alcohol (PVA) vs. sodium cholate), were intravenously administered in before- or early-onset treatment schedules in a rat EAN model. NP treatment mitigated distinctly the clinical severity of EAN as compared to the P2-peptide control group (P2) in all treatments and reduced the trafficking of inflammatory monocytes at inflammatory loci and diverted them towards the spleen. Therapeutic treatment with NPs reduced the expression of proinflammatory markers (CD68 (P2: 34.8 ± 6.6 vs. NP: 11.9 ± 2.3), IL-1β (P2: 18.3 ± 0.8 vs. NP: 5.8 ± 2.2), TNF-α (P2: 23.5 ± 3.7 vs. NP: 8.3 ± 1.7) and elevated the expression levels of anti-inflammatory markers CD163 (P2: 19.7 ± 3.0 vs. NP: 41.1 ± 6.5; all for NP-PVA of 130 nm; relative to healthy control). These results highlight the therapeutic potential of such cargo-free NPs in treating EAN, which would be easily translatable into clinical use due to their well-known low-toxicity profile.